Pharmaceutical compositions comprising polymeric binders with non-hydrolysable covalent bonds and their use in treating celiac disease

ABSTRACT

A pharmaceutical composition comprising a polymeric binder including a high molecular weight synthetic polymer having a backbone constituted of non hydrolysable covalent bonds, said polymer being able to form electrostatic bonds at a pH lower than the isoelectric point of gluten and peptides derived from the degradation of gluten, and being able to bind to gluten or peptides derived from the degradation of gluten in the gastrointestinal tract, and a pharmaceutically acceptable carrier. Methods of using the polymeric binder for binding gluten or a peptide derived from the degradation of gluten, for decreasing the degradation of gluten into toxic peptides or for decreasing interaction of gluten or peptides derived from the degradation of gluten with the gastrointestinal mucosa.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority on U.S. provisional application No.60/735,820, filed on Nov. 14, 2005. All documents above are herein intheir entirety by reference.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions comprisingpolymeric binders, and methods of use thereof. More specifically, thepresent invention is concerned with non-digestible synthetic polymersfor binding gluten or gliadin and/or -peptides derived from thedegradation of gluten or gliadin and methods of use thereof.

BACKGROUND OF THE INVENTION

Celiac disease, also known as gluten intolerance is a syndromecharacterized by damage to the small intestinal mucosa, followingexposure to either the gliadin fraction of wheat gluten or similaralcohol soluble proteins (prolamines) of barley and rye in geneticallysusceptible subjects. Celiac disease is a common autoimmune disorderthat has genetic, environmental and immunologic components. The diseaseis closely associated with genes that code for human leukocyte antigensDQ2 and DQ8 (1). A 33-mer fragment of α-gliadin was identified that hasseveral characteristics suggesting it is a possible initiator of theinflammatory response to gluten in celiac disease patients (2).

Symptoms of celiac disease can range from mild weakness, bone pain, andaphthous stomatitis to chronic diarrhea, abdominal bloating, andprogressive weight loss (3). Because of the broad range of symptoms,celiac disease presence can be difficult to diagnose. Those affectedsuffer damage to the villi (shortening and villous flattening) in thelamina propria and crypt regions of their intestine (3). Furthermore,gastrointestinal carcinoma or lymphoma develops in up to 15 percent ofpatients with untreated or refractory celiac disease (4). A gluten-freediet can prevent almost all complications of the disease (5). Such adiet involves avoiding all products that contain wheat, rye, barley, orany of their derivatives. This is a difficult task as many hiddensources of gluten can be found in the ingredients of many processedfoods.

Until now, aside from excluding gluten-containing foods from their diet,no pharmacological treatment is available for celiac patients.Surprisingly, relatively few treatment strategies are currently beingexplored. Approaches based on the tolerance of antibody and T-cellmediated response to the gliadin toxic peptides or on the development ofanti-IL-15 neutralizing antibodies blocking the IL-15 mediated changesin the small intestinal mucosa are under investigation (6). A promisingavenue lies in the discovery of exogenous enzymes, which could rapidlydegrade toxic peptides in situ (7). However, the high cost associated tolarge-scale enzyme production and possible loss of activity after oraladministration are potential constraints to their commercialization.Complementary strategies aiming to interfere with activation ofgluten-reactive T cells include the inhibition of binding of glutenpeptides to human leukocyte antigen (HLA) 002 (or DQ8). The crucial roleof HLA in celiac disease development makes it an obvious target fortherapeutic intervention. The recently solved X-ray crystal structure ofHLA-DQ2 complexed with a deaminated gluten peptide has providedimportant information for the development of an HLA-DQ2-blockingcompound (8). Zonulin antagonists have also been suggested as therapyfor celiac disease. Zonulin is a protein involved in the regulation ofintercellular tight junctions in the small intestine. Its expression hasbeen shown to increase during the acute phase of celiac disease, aclinical condition in which the intestinal permeability is increased(9).

The development of grains that have low or no content of immunotoxicsequences, but with reasonable baking quality, has also beeninvestigated. Such grains can potentially be developed by selectivebreeding of ancient wheat varieties (10), by transgenic technologyinvolving mutation of sequences giving rise to immunostimulatorysequences (11) or by incorporation of nontoxic gluten genes intoharmless organisms such as rice (12). Although these grains aretechnically challenging to engineer, and there is a possibility thatcross-pollination with gluten-containing grains might lead toreintroduction of immunotoxic sequences, the availability of such grainscould give patients with celiac disease a nutritionally better diet.

Polymeric Binders

A number of polymeric binders have been used for treating or preventingcertain diseases.

The classic example of a polymeric binder is cholestyramine, a cationicresin that sequesters biliary acids in the gut and consequently lowerscholesterol blood levels. Recently, sevelamer hydrochloride, a novelaluminum and calcium-free polymeric phosphate binder with negligibleside effects has been commercialized for the treatment ofhyperphosphatemia in patients on dialysis. Perhaps the most interestingdiscovery in this field is an anionic high-molecular weight polymer,GT160-246, which was shown to neutralize Clostridium difficile toxin Aactivity both in vitro and in vivo (13). This endotoxin is the mostcommonly identified cause of infectious nosocomial diarrhea. GT160-246offers a promising and safe nonantimicrobial approach to the treatmentand prevention of C. difficile colitis in humans.

The idea that high molecular weight polymers could be of potential usein celiac disease stemmed from a study of Auricchio et al. (14), whichdemonstrated that mannan (mannose homopolysaccharide) andacetylglucosamine oligomers exhibited a protective effect on intestinalmucosa specimens of patients with active celiac disease (14). Thesefindings suggest that the agglutinating and toxic peptides are bound bythese carbohydrates. Secundo et al. (26) explored the effect of an otherpolysaccharide, dextrin on the secondary structure of gliadins andhypothesized that dextrin might be used to prepare non toxic foodderivatives for patients suffering from celiac disease. Despite theseinteresting preliminary data, no further investigations were carried outto confirm those findings in vivo. The main drawback of naturalcarbohydrates is their degradability under in vivo conditions whichwould make them inactive in situ.

The present invention seeks to meet these needs and other needs.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

More specifically, in accordance with an aspect of the presentinvention, there is provided a pharmaceutical composition comprising apolymeric binder including a high molecular weight synthetic polymerhaving a backbone constituted of non hydrolysable covalent bonds, saidpolymer being able to form electrostatic bonds at a pH lower than theisoelectric point of gluten and peptides derived from the degradation ofgluten, and being able to bind to gluten or peptides derived from thedegradation of gluten in the gastrointestinal tract, and apharmaceutically acceptable carrier.

In a specific embodiment of the pharmaceutical composition, thepolymeric binder is able to form hydrophobic interactions with gluten orpeptides derived from the degradation of gluten. In an other specificembodiment of the pharmaceutical composition, the polymeric binder isable to form hydrogen bonds. In an other specific embodiment of thepharmaceutical composition, the polymeric binder is able to specificallybind to gluten or peptides derived from the degradation of gluten in thegastrointestinal tract. In an other specific embodiment of thepharmaceutical composition, the polymeric binder is able to bind togluten or peptides derived from the degradation of gluten in theintestinal tract. In an other specific embodiment of the pharmaceuticalcomposition, the polymeric binder is a copolymer of hydroxyethylmethacrylate (HEMA) and 4-styrene sulfonic acid sodium salt hydrate(SStNa). In an other specific embodiment of the pharmaceuticalcomposition, the polymeric binder is a polymer of 4-styrene sulfonicacid sodium salt hydrate (SStNa). In an other specific embodiment of thepharmaceutical composition, the polymeric binder is a polymer ofsulfopropyl methacrylate potassium salt (SPMAK).

In an other specific embodiment of the pharmaceutical composition, thepolymeric binder is linear. In an other specific embodiment of thepharmaceutical composition, the polymeric binder is star-shaped. In another specific embodiment of the pharmaceutical composition, thepolymeric binder is a 3 to 18-arm star-shaped copolymer. In an otherspecific embodiment of the pharmaceutical composition, the polymericbinder is a 5 to 18-arm star-shaped copolymer. In an other specificembodiment of the pharmaceutical composition, the polymeric binder is a5-arm star-shaped copolymer. In an other specific embodiment of thepharmaceutical composition, the polymeric binder is a 8-arm star-shapedcopolymer. In an other specific embodiment of the pharmaceuticalcomposition, the polymeric binder is a 18-arm star-shaped copolymer. Inan other specific embodiment of the pharmaceutical composition, thepolymeric binder is a copolymer of HEMA and SStNa and has a HEMA/SStNamolar percentage ratio between about 93.5/6.5 and about 1/99. In another specific embodiment of the pharmaceutical composition, thecopolymer is linear HEMA/SStNa (51.5/48.5 mol %). In an other specificembodiment of the pharmaceutical composition, the copolymer is linearHEMA/SStNa (43/57 mol %). In an other specific embodiment of thepharmaceutical composition, the copolymer has a HEMA/SPMAK molarpercentage ratio between about 93.5/6.5 and about 1/99%). In an otherspecific embodiment of the pharmaceutical composition, the copolymer hasa HEMAISPMAK molar percentage ratio between about 86/14 and about 1/99.

In an other specific embodiment of the pharmaceutical composition, thecopolymer is linear HEMA/SPMAK (45/55 mol %).

In an other specific embodiment, the pharmaceutical composition of thepresent invention further comprises a zonulin antagonist or an HLA DQ2inhibitor.

In accordance with an other aspect of the present invention, there isprovided a method of using the polymeric binder of the present inventioncomprising administering to a patient suffering from celiac disease apharmaceutically effective amount of said polymeric binder.

In a specific embodiment, the method of the present invention is forbinding gluten or a peptide derived from the degradation of gluten inthe patient.

In an other specific embodiment, the method of the present invention isfor decreasing the degradation of gluten into toxic peptides in thepatient.

In an other specific embodiment, the method of the present invention isfor decreasing interaction of gluten or peptides derived from thedegradation of gluten with the gastrointestinal mucosa of the patient.

In an other specific embodiment of the method of the present invention,said administration is performed before or during a gluten-containingmeal of said patient. In an other specific embodiment of the method ofthe present invention, said administration is performed after agluten-containing meal of said patient.

In accordance with an other aspect of the present invention, there isprovided a use of the polymeric binder of the present invention in thepreparation of a medicament.

In accordance with an other aspect of the present invention, there isprovided a use of the polymeric binder of the present invention forbinding gluten or a peptide derived from the degradation of gluten inthe gastrointestinal tract of a patient in need thereof.

In accordance with an other aspect of the present invention, there isprovided a use of the polymeric binder of the present invention in thepreparation of a medicament for binding gluten or a peptide derived fromthe degradation of gluten in the gastrointestinal tract of a patient inneed thereof.

In accordance with an other aspect of the present invention, there isprovided a use of the polymeric binder of the present invention fordecreasing interaction of gluten or peptides derived from thedegradation of gluten with the gastrointestinal mucosa of a patient inneed thereof.

In accordance with an other aspect of the present invention, there isprovided a use of the polymeric binder of the present invention in thepreparation of a medicament for decreasing interaction of gluten orpeptides derived from the degradation of gluten with thegastrointestinal mucosa of a patient in need thereof.

In accordance with an other aspect of the present invention, there isprovided a use of the polymeric binder of the present invention fordecreasing degradation of gluten into toxic peptides in thegastrointestinal tract of a patient in need thereof.

In accordance with an other aspect of the present invention, there isprovided a use of the polymeric binder of the present invention in thepreparation of a medicament for decreasing the degradation of gluteninto toxic peptides in the gastrointestinal tract of a patient in needthereof.

In a specific embodiment of the use of the present invention, thepatient suffers from celiac disease.

In accordance with yet an other aspect of the present invention, thereis provided food comprising the polymeric binder of the presentinvention.

In a specific embodiment of the food of the present invention, said foodis a gluten-containing food. In a specific embodiment of the food of thepresent invention, said food is bread.

In accordance with yet an other aspect of the present invention, thereis provided a method of using the food of the present invention,comprising administering said food to a patient suffering from celiacdisease during the patient's meal. In a specific embodiment, the methodof the present invention is for binding gluten or a peptide derived fromthe degradation of gluten contained in the meal of the patient. In another specific embodiment, the method of the present invention is fordecreasing the degradation into toxic peptides of gluten contained inthe meal of the patient. In a specific embodiment, the method of thepresent invention is for decreasing interaction of gluten or peptidesderived from the degradation of gluten with the gastrointestinal mucosaof the patient.

In accordance with yet an other aspect of the present invention, thereis provided a method of making food for a patient suffering from celiacdisease, comprising incorporating into said food the polymeric binder ofthe present invention. In a specific embodiment of the method of thepresent invention, said food is a gluten-containing food.

The present invention concerns a high molecular weight inert andnon-absorbable polymeric binder, which for use to adsorb gluten and/orits degradation products. Such a system will help prevent or decreasegluten's deleterious effects on the gastrointestinal mucosa. Withoutbeing so limited, it is hypothesized that peptide binding to the polymerhas two effects. First, the enzymatic degradation and generation oftoxic fragments is slowed down by gluten and/or by its degradationproduct's adsorption on an inert support. Second, complexation with ahigh molecular weight polymer decreases peptide absorption and thesubsequent immune response. This system thus provides a preventionadjuvant for patients faced with situations where the absence of glutenresidues cannot be ascertained or when gluten free meals are notavailable.

Although specific non-digestible synthetic polymers are presentedherein, the invention is not so limited. As used herein, the terms“non-digestible” when used to qualify the polymers of the presentinvention, is meant to refer to a polymer having a backbone constitutedof non hydrolysable covalent bonds. It is believed that a person ofordinary skill in the art may easily identify other non-digestiblesynthetic polymers that can be used in accordance with the presentinvention. Similarly, the polymers specifically described herein can beoptimized to maximize their affinity towards gluten and its degradationproducts and minimize their binding to other proteins. Of course, acertain proportion of these proteins/peptides will escape adsorptiononto the polymers of the present invention but it has been suggestedthat a daily intake of gliadin of 4-14 mg does not causesmall-intestinal mucosal damage in celiac patient (15). Such a systemwould certainly not replace a gluten free diet as main treatment.However, it could be used occasionally as a prevention adjuvant whenpatients face situations where absence of gluten residues cannot beascertained or when gluten free meals are not available.

The polymeric binders of the present invention may advantageously reducethe oral absorption of gluten and peptides derived thereof. Thesepolymeric binders act in the gastrointestinal tract without beingabsorbed into the bloodstream, thereby minimizing the potential foradverse effects caused by the polymer itself. At a pH lower than theisoelectric point of gluten and peptides derived thereof, the polymericbinders are negatively charged while these proteins and peptides arepositively charged allowing for the formation of electrostaticinteractions. These polymeric binders also may also form hydrophobicinteractions with these proteins and peptides. In specific embodiments,the polymeric binders of the present invention also have an ability toform hydrogen bonds. Although this last characteristic may be desirable,it was shown not to be essential since it certain polymers of thepresent invention, e.g. homopolymer of sulfopropyl methacrylatepotassium salt (SPMAK), that do not possess this characteristic werefound to be able to bind to gluten. Without being so limited, suchpolymeric binders can be synthesized with monomers presented in Table 1below. People of ordinary skill in the art may select combinations ofone or more of these (or other) monomers to form polymeric binders ofthe present invention:

TABLE 1 Styrene derivatives: Styrene sulfonate. Styrene sulfate. Styrenesulfanilate. Sulfophenyl alanine. Tyrosine sulfate. Sulfophenethylacrylamide. Sulfophenethyl methacrylamide. Vinyl naphthalene sulfonate.Vinylnaphthalene sulfate. Vinylbiphenyl sulfonate. Vinylbiphenylsulfate. Anethole sulfonate. Styrenes with crown ether moieties.Styrenes substituted with N,N-dialkylamido groups. 4-methoxystyrene.4-(2-(N,N-dimethylamino)ethyl) styrene. 4-(2-(N,N-dimethylamino)methyl)styrene 4-(2-(N,N-diethylamino) ethyl) styrene4-bis(N,N-diethylamino)phosphino-a-methyl styrene. 4-vinylphenol.3-vinylcatechol. 4-vinylacetophenone. 4-vinylbenzoic acid.3-vinylbenzoic acid 2-(4-vinylphenyl)-1,3-dioxolane.2-(4-vinylphenyl)-1,3-dioxane.4-dimethoxymethylstyrene-(4-vinylbenzaldehyde dimethylacetal).2-(2-vinylphenyl)-1,3-dioxolane. 2-(3-vinylphenyl)-1,3-dioxolane.1-(4-vinylphenyl)-4-methyl-2,6,7-trioxabicyclo[2.2.2]octane.4-(2-hydroxyethyl) styrene 4-(3-hydroxypropyl) styrene4-{[4-(4-vinylphenyl)butoxy]methyl}-1-methyl-2,6,7-trioxabicyclo[2.2.2]octane. 4-vinylthiophenol. 4-(2-mercaptoethyl)styrene. 2-(4-vinylphenyl)-2-oxazoline.N,N-diethyl-4-vinylbenzenesulfonamide.N-methyl-N′-[(4-vinylphenyl)sulfonyl]piperazine. 4-aminostyrene.3-aminostyrene 4-aminomethylstyrene. 3-aminomethylstyrene.4-(2-aminoethyl)styrene. Styrene bearing hydroxyl group(s):(p-Vinylbenzamido)-β-chitobiose. (p-vinylbenzamido)-β-lactose.N-(p-vinylbenzyl)-L-gulonamide. N-(p-vinylbenzyl)-6-D-glucaramide.N-(p-vinylbenzyl)-6-D-glucaramid-1-ate. 4-Acrylamidophenyl-β-lactoside.N-(p-vinylbenzyl)-D-glucoronamide.4-vinylbenzyl-D-gluco(D-manno)hexitol.p-[2-[N-(p-vinylbenzyl)carbamoyl]ethyl]phenyl α-D-mannopyranoside.p-[2-[N-(p-vinylbenzyl)carbamoyl]ethyl]phenyl β-D-mannopyranoside.N-(p-vinylbenzyl)-5[O-β-D-galactopyranosyl- (1→4)]-D-gluconamide.α-mannopyranoside. β-mannopyranoside. Acrylic monomers: Glycidylacrylate. 2-Hydroxyethyl acrylate. 2-Hydroxyethyl methacrylate.Hydroxypropyl methacrylate. 2-(N,N-Dimethylamino)ethyl methacrylate.2-(N,N-Diethylamino)ethyl methacylate 3-Sulfopropyl methacrylate.Tetrahydropyranyl methacrylate. Benzyl methacrylate. 2-gluconamidoethylmethacrylate. 2-lactobionamidoethyl methacrylate.2-(2′,3′,4′,6′-tetra-O-acetyl-β-D-glucopyranosyloxy) ethyl acrylate.(4,5-dihydroxy-6-hydroxymethyl-3- methylcarboxamidotetrahydro-2H-2-pyranyloxy)ethyl acrylate. Sulfated monomers: Vinyl sulfate. Propenesulfate. Butene sulfate. Pentene sulfate. Hexene sulfate. Heptenesulfate. Octene sulfate. Nonene sulfate. Decene sulfate. Undecenesulfate. Dodecene sulfate. Sulfonated monomers: Vinyl sulfonate. Propenesulfonate. Butene sulfonate. Pentene sulfonate. Hexene sulfonate.Heptene sulfonate. Octene sulfonate. Nonene sulfonate. Decene sulfonate.Undecene sulfonate. Dodecene sulfonate. Phosphated monomers: Vinylphosphate. Propene phosphate. Butene phosphate. Pentene phosphate.Hexene phosphate. Heptene phosphate. Octene phosphate. Nonene phosphate.Decene phosphate. Undecene phosphate. Dodecene phosphate. Others: Maleicanhydride. N-acryloylated 3′-sulfo-Lewisx-Glc monomer. α-sialosideacrylamide. N-vinylpyridine. N-vinylpyrrolidinone. Vinyl imidazole.1,3-Dimethyl-2-(4-vinylphenyl)imidazolidine.3-(N-acryloylamino)propyl-O-(β-D-galactopyranosyl)- (1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside.6-(N-acryloylamino)hexyl-O-(β-D-galactopyranosyl)-(1→4)- 2-acetamido-2-deoxy-β-D-glucopyranoside. 3-(N-acryloylamino)propyl2-acetamido-2-deoxy-β-D- glucopyranoside. 6-(N-acryloylamino)hexyl2-acetamido-2-deoxy-~-D- glucopyranoside. n-pentenylβ-D-galactopyranoside.n-pentenyl-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→4)-2-acetamido-2- deoxy-β-D-glucopyranoside.n-pentenyl-O-(β-D-galactopyranosyl)-(1→4)-2-acetam ido- 2-deoxy-β-D-glucopyranoside. n-pentenyl-O-(β-D-galactopyranosyl)-(1→4)-β- D-giucopyranoside. n-pentenyl-O-(β-D-galactopyranosyl)-(1→4)-[O-(α-L-fucopyranosyl)-(1→3)]-2- acetamido-2-deoxy-β-D-glucopyranoside.n-pentenyl-O-(β-D-galactopyranosyl)-(1→6)-2- acetamido-2-deoxy-β-D-glucopyranoside. n-pentenyl-O-(β-D-galactopyranosyl)-(1→3)-2-acetamido-2-deoxy-β-D- glucopyranoside.n-Alkenyl-2-acetamido-2-deoxy-α-D-glucopyranosides (and sulfatedderivatives); 2-N-acryloyl-aminoethoxyl 4-O-(β-D-galactopyranosyl)-β-D-glucopyranoside (and sulfated derivatives).N-maleicamido-2-deoxy-glucose sodium salt.N-maleicamido-1-deoxy-lactitol sodium salt. Fucose-7-oxanorbornenederivative. C-Glc-7-oxanorbornene derivative. C-Man-7-oxanorbornenederivative. Unsymmetrical glucose containing 7-oxanorbornene derivative.O-Glc-7-oxanorbornene derivative. O-Man-7-oxanorbornene derivative.Unsymmetrical mannose containing 7-oxanorbornene derivative. O-Mannorbornene derivative. sugar derivatized poly(7-oxanorbornene)s. sugarderivatized poly(norbornene)s.

While the polymeric binders of the present invention have backboneconstituted of non hydrolysable covalent bonds, they may also compriseside chains containing hydrolysable covalent bonds.

As used herein the term “gluten” refers to a protein group found invarious cereals. Gluten can be fractioned into the ethanol-solubleprolamines and ethanol-insoluble glutenins. Alcohol-soluble prolaminesfrom wheat, rye, barley and possibly oats are toxic in celiac patients.A common feature of the wheat prolamine is a high content of glutamine(>30%) and proline (>15%). The wheat prolamines are subdivided into al˜,y and ro gliadins containing similar or repetitive glutamine andproline-rich peptide epitopes that appear to be responsible for theobserved toxicity of gluten.

As used herein, the term “peptide derived from the degradation ofgluten” refers to any peptide derived from the degradation of glutenthat would desirably bind to the polymers of the present invention aftergluten intake. Without being so limited it includes all peptides listedin Ciccocioppo (23).

As used herein, the term “high molecular weight polymer” refers to apolymer having a molecular weight comprised between 5,000 and 5,000,000g/mol.

As used herein, the term “pharmaceutically acceptable carrier” refers toa solution, suspension, emulsion, tablet or capsule prepared withcommonly used excipients such as those described in Modern Pharmaceutics(27).

As used herein, the term “pharmaceutically effective amount” of apolymer of the present invention refers to an amount that is effectivefor decreasing interaction of gluten or peptides derived from thedegradation of gluten with the gastrointestinal mucosa after glutenintake of a patient in need thereof. Without being so limited, theeffective amount of the polymer of the present invention may be fromabout 200 mg up to about 15 g per day (e.g., 200 mg; 250 mg; 300 mg; 500mg; 750 mg; 1 g; 1.5 g; 2 g; 2.5 g; 3 g, 5 g; 7.5 g).

As used herein, the term “specifically binds” in the expression “polymerbinder that specifically binds to gluten or peptides derived from thedegradation of gluten” refers to the ability of the polymer to bind morein the gastrointestinal tract to proteins or peptides derived from foodintake that are hydrophobic such as gluten and peptides derived thereofthan they bind to other food proteins such as casein and/or albumin.

The present invention encompasses linear and star-shaped polymers.Star-shaped polymers according to specific embodiments of the presentinvention have 3 to 18 arms.

As used herein the term “patient in need thereof” refers to a humanaffected by celiac disease that is eating or has eaten agluten-containing meal.

The articles “a,” “an” and “the” are used herein to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle.

The term “including” and “comprising” are used herein to mean, and reused interchangeably with, the phrases “including but not limited to”and “comprising but not limited to”.

The term “such as” is used herein to mean, and is used interchangeablywith, the phrase “such as but not limited to”.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 presents the chemical structures of linear and multifunctionalATRP initiators used to synthesize polymers described herein. (i)PEG-dibromo macroinitiator; (ii) 1,2,3,4,6-penta-O-isobutyrylbromide-R-D-Glucose; (iii) Octadeca-O-isobutyryl bromide-R-cyclodextrin;(iv) Octa-O-isobutyryl bromide-sucrose;

FIG. 2 presents the SOS-PAGE of the binding of albumin and α-gliadinwith poly(HEMA-co-SStNa) (Example 10) at pH 6.8 in triplicate: (A)protein standards; (8) albumin and a-gliadin mixture; (C) mixture ofalbumin (40 mg/L), α-gliadin (40 mg/L) and poly(HEMA-co-SStNa) (160mg/L);

FIG. 3 is a binding profile of linear poly(HEMA-co-SStNa) to gliadin,albumin, and casein at pH 1.2 and 6.8, wherein each point corresponds tothe polymer of each of Examples 5 to 10 and 12-13;

FIG. 4 is a binding profile of linear poly(HEMA-co-SPMAK) to gliadin andalbumin at pH 1.2 and 6.8 wherein each point corresponds to the polymerof each of Examples 17 to 21;

FIG. 5 graphically presents the polymer structure effect on the bindingof gliadin at neutral pH (SStNa=25-31 mol %; see Examples 9, 14, 15 and16);

FIG. 6 graphically presents the polymer structure effect on the bindingof gliadin at neutral pH (SPMAK=16-19 mol %; see Examples 13, 22, 23 and24);

FIG. 7 presents the effect of a polymer of the invention on gliadindigestion under simulated intestinal conditions. Comparative HPLCprofiles of gliadin digested with pepsin, trypsin and chymotrypsin (PTC)in absence (a) and presence (b) of polymer. Chromatogram (c) correspondsto intact α-gliadin;

FIG. 8 presents the variation of the transepithelial electric resistance(TEER) of a Caco-2 monolayer following incubation with solutions of PEG(Mn: 35,000; open circles), PVP (Mw: 58,000; closed squares),poly(HEMA-co-SStNa) (Example 10; open triangles) and complete medium(closed stars) as a control. Cells were maintained in DMEM cell culturemedia supplemented with 10% FBS. The polymer concentration was fixed at1 g/L; and

FIG. 9 presents a binding profile of two linear poly(HEMA-co-SStNa)containing about 50% SStNa and having two different molecular weights(Examples 10 and 11) to gliadin and albumin at pH 1.2 and 6.8.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Materials

α-Gliadin was kindly supplied by the Institut National de la RechercheAgronomique, (Nantes, France). It was purified from soft wheat asdescribed by Popineau et al. (16-21). Briefly, after extraction of crudegliadin from gluten (isolated from flour), gliadin subgroups wereseparated and purified successively by ion exchange chromatography, sizeexclusion chromatography and finally hydrophobic interactionchromatography.

Bovine albumin was purchased from Serological Proteins (Kankakee, Ill.).α-Casein (from bovine milk), SStNa, HEMA, SPMAK, R-D-glucose,α-cyclodextrin hydrate, sucrose (98%), poly(ethylene glycol) (PEG)(M_(n) 2000), 2-bromoisobutyryl bromide, copper bromide Cu(I)Br and2,2′dipyridyl were all purchased from Sigma-Aldrich (St Louis, Mo.) andused as received. Eppendorff tubes, pipette tips and 96-well plates(Maximum Recovery) were provided from Axygen Scientific (Union City,Calif.).

Synthesis of the Initiators

Atom transfer radical polymerization (ATRP) initiators (FIG. 1) wereprepared from PEG, R-D-glucose, sucrose and a-cyclodextrin. The bromidefunctionalization of the last three molecules was achieved by theapproach described by Stenzel-Rosenbaum and co-workers (22).

The present invention is illustrated in further details by the followingnon-limiting examples.

Example 1 Synthesis of PEG Dibromomacroinitiator (i)

A solution of HO-PEG-OH (M_(n) 2000, 10 g, 5 mmol) and triethylamine (10g, 0.1 mol) in 70 mL of anhydrous toluene was slightly cooled in anice-water bath. Then, 2-bromoisobutyryl bromide (4.91 mL, 0.04 mol) wasslowly added to the reaction mixture. The solution was warmed to roomtemperature and stirred for 48 h. The mixture was filtered, half of thesolvent was evaporated, and the PEG macroinitiator was precipitated incold diethyl ether (FIG. 1 (i)).

Yield: 90%, after precipitation. White solid. ¹H NMR (δ, ppm, CDCl₃):3.50 (188H), 1.80 (12H, s).

Example 2 Synthesis of 1,2,3,4,6-Penta-O-isobutyryl bromide-R-D-glucose(ii)

2-bromoisobutyryl bromide (50 g, 0.22 mol) was slowly added to asolution of R-D-glucose (5.0 g, 0.028 mol) in an anhydrous mixture ofchloroform (100 mL) and pyridine (50 mL). The solution was refluxed for3 h while maintaining a dry atmosphere and then stirred at roomtemperature for a further 12 h. It was then washed successively withice-cold water, NaOH (0.1 M), and water and dried over anhydrous MgSO₄.The crude product was recrystallized from methanol to yield whitecrystals (FIG. 1 (ii)).

Yield: 70%. ¹H NMR (CDCl₃): 1.85-2.04 (m, 30H, H-7), 6.42 (d, 1H, H-1),5.25 (dd, 1H, H-2), 5.69 (t, 1H, H-3), 5.35 (t, 1H, H-4), 4.38 (m, 3H,H-5/6).

Example 3 Synthesis of Octadeca-O-isobutyryl Bromide-R-cyclodextrin(iii)

Octadeca-O-isobutyryl bromide-R-cyclodextrin was synthesized by the slowaddition of 2-bromoisobutyryl bromide (50 g, 0.22 mol) to a solution ofR-cyclodextrin (5.0 g, 0.005 mol) in anhydrous pyridine (150 mL). Thesolution was stirred for 24 h under a dry atmosphere at roomtemperature. It was then washed with ice-cold water, NaOH (0.1 M), andwater, respectively, prior to drying over anhydrous MgSO₄. The crudeproduct was recrystallized from methanol/H₂O (3:1, v/v) to yield whitecrystals (FIG. 1 (iii)).

Yield: 55%. ¹H NMR (CDCl₃): 1.95 (m, 108H, H-7), 5.84 (d, 12H, H-1),4.46 (dd, 6H, H-2), 5.7 (m, 6H, H-3), 5.13/5.38 (t/dd, 6H, H-4), 4.78(dd, 6H, H-5), 4.45 (m, 6H, H-6).

Example 4 Synthesis of Octa-O-isobutyryl bromide-sucrose (iv)

Octa-O-isobutyryl bromide sucrose was synthesized by the slow additionof 2-bromoisobutyryl bromide (50 g, 0.22 mol) to a solution of sucrose(5.0 g, 0.014 mol) in anhydrous pyridine (150 mL). The solution wasstirred for 24 h under a dry atmosphere at room temperature. It was thenwashed with ice-cold water, NaOH (0.1 M), and water, prior to dryingover anhydrous MgSO₄. The crude product was recrystallized frommethanol/H₂O (3:1 v/v) to yield white crystals (FIG. 1 (vi)).

Yield: 50%. ¹H NMR (CDCl₃): 1.99 (m, 48H, H-7), 4.15 (d, 1H, H-5′), 4.46(m, 5H, H-6′/1′/5), 4.68 (dt, 2H, H-6), 4.81 (d, 1H, H-3′), 5.13 (dd,1H, H-2), 5.38 (t, 1H, H-4′), 5.67 (t, 1H, H-4), 5.76 (t, 1H, H-3), 5.85(d, 1H, H-1).

Example 5 Synthesis of Linear Hydroxyethyl Methacrylate (HEMA)/4-StyreneSulfonic Acid Sodium Salt Hydrate (SStNA) Copolymer (93.5/6.5 mol %After Purification)

The ATRP initiator i (FIG. 1) (50 mg), SStNa (0.375 g) and HEMA (7.12 g)were dissolved in 46 mL of a methanol/water (1/4) mixture and degassedunder argon for 15 min. Bpy (20.28 mg), Cu(I)Br (7.2 mg) and Cu(II)Br₂(3.35 mg) were then added under stirring at 20° C., After 24 h, thesolution was exposed to air and the dark-brown solution turned to blue,indicating oxidation of Cu(I) to Cu(II). The polymer was purified bypassing the methanol/water solution through a silica gel column whichremoved the Cu(II) catalyst. The polymers were dialyzed (Spectra/Por™no. 1, MW cutoff 6000-8000 Spectrum Laboratories, Rancho Dominguez,Calif.) against water for 48 h and then freeze-dried until use.M_(w)=318700 g/mol; M_(w)/M_(n)=2.54.

Example 6 Synthesis of Linear Hydroxyethyl Methacrylate/4-StyreneSulfonic Acid Sodium Salt Hydrate Copolymer (90.3/9.7 mol % AfterPurification)

The ATRP initiator i (FIG. 1) (50 mg), SStNa (0.375 g) and HEMA (7.12 g)were dissolved in 46 mL of a methanol/water (1/4) mixture and degassedunder argon for 15 min. Bpy (21.84 mg), Cu(I)Br (7.2 mg) and Cu(I)Br₂(4.48 mg) were then added under stirring at 20° C. After 24 h, thesolution was exposed to air and the polymer was purified as reported inExample 5. M_(w)=331 528, M_(w)/M_(n)=2.9

Example 7 Synthesis of Linear Hydroxyethyl Methacrylate/4-StyreneSulfonic Acid Sodium Salt Hydrate Copolymer (87.8/12.2 mol % AfterPurification)

The ATRP initiator i (FIG. 1) (50 mg), SStNa (0.75 g) and HEMA (6.747 g)were dissolved in 46 mL of a methanol/water (1/4) mixture and degassedunder argon for 15 min. Bpy (15.6 mg) and Cu(I)Br (7.2 mg) were thenadded under stirring at 20° C. After 24 h, the solution was exposed toair and the polymer was purified as reported in Example 5. M_(w)=283 600g/mol; M_(w)/M_(n)=2.57.

Example 8 Synthesis of Linear Hydroxyethyl Methacrylate/4-StyreneSulfonic Acid Sodium Salt Hydrate Copolymer (82.4/17.6 mol % AfterPurification)

The ATRP initiator i (FIG. 1) (50 mg), SStNa (1.125 g) and HEMA (6.426g) were dissolved in 46 mL of a methanol/water (1/4) mixture anddegassed under argon for 15 min. Bpy (15.6 mg) and Cu(I)Cl (5 mg) werethen added under stirring at 20° C. After 24 h, the solution was exposedto air and the polymer was purified as reported in Example 5. M_(w)=275500 g/mol; M_(w)/M_(n)=2.5.

Example 9 Synthesis of Linear Hydroxyethyl Methacrylate/4.StyreneSulfonic Acid Sodium Salt Hydrate Copolymer (69/31 mol % AfterPurification)

The ATRP initiator i (FIG. 1) (50 mg), SStNa (1.5 g) and HEMA (5.99 g)were dissolved in 46 mL of a methanol/water (1/4) mixture and degassedunder argon for 15 min. Bpy (15.6 mg) and Cu(I)Cl (5 mg) were then addedunder stirring at 20° C. After 24 h, the solution was exposed to air andthe polymer was purified as reported in Example 5. M_(w)=NA;M_(w)/M_(n)=NA. Mn_((NMR))=58 100 g/mol.

Example 10 Synthesis of Linear Hydroxyethyl Methacrylate/4-StyreneSulfonic Acid Sodium Salt Hydrate Copolymer (51.5/48.5 mol % AfterPurification)

The ATRP initiator i (FIG. 1) (50 mg), SStNa (3.2 g) and HEMA (3.95 g)were dissolved in 46 mL of a methanol/water (1/4) mixture and degassedunder argon for 15 min. Bpy (15.6 mg) and Cu(I)Br (7.2 mg) were thenadded under stirring at 20° C. After 24 h, the solution was exposed toair and the polymer was purified as reported in Example 5. M_(w)=122 000g/mol; M_(w)/M_(n)=2.23.

Example 11 Synthesis of Linear Hydroxyethyl Methacrylate/4.StyreneSulfonic Acid Sodium Salt Hydrate Copolymer (43/57 mol % AfterPurification)

The ATRP initiator i (FIG. 1) (50 mg), SStNa (2.4 g) and HEMA (1 g) weredissolved in 23 mL of a methanol/water (1/4) mixture and degassed underargon for 15 min. Bpy (15.6 mg) and Cu(I)Br (7.2 mg) were then addedunder stirring at 20° C. After 24 h, the solution was exposed to air andthe polymer was purified as reported in Example 5. Mn_((NMR))=55 000g/mol.

Example 12 Synthesis of Linear Hydroxyethyl Methacrylate/4-StyreneSulfonic Acid Sodium Salt Hydrate Copolymer (28/72 mol % AfterPurification)

The ATRP initiator i (FIG. 1) (50 mg), SStNa (4.8 g) and HEMA (1.975 g)were dissolved in 46 mL of a methanol/water (1/4) mixture and degassedunder argon for 15 min. Bpy (15.6 mg) and Cu(I)Br (7.2 mg) were thenadded under stirring at 20° C. After 24 h, the solution was exposed toair and the polymer was purified as reported in Example 5. M_(w)=65 200g/mol; M_(w)/M_(n)=1.95.

Example 13 Synthesis of Linear Poly(4-Styrene Sulfonic Acid Sodium SaltHydrate)

The ATRP initiator i (FIG. 1) (50 mg) and SStNa (6.4 g) were dissolvedin 46 mL of water and degassed under argon for 15 min. Bpy (15.6 mg) andCu(I)Br (7.2 mg) were then added under stirring at 20° C. After 24 h,the solution was exposed to air and the polymer was purified as reportedin Example 5. M_(w)=NA; M_(w)/M_(n)=NA. M_(n (NMR))=20 000 g/mol.

Example 14 Synthesis of Linear Poly(4.Styrene Sulfonic Acid Sodium SaltHydrate)

The ATRP initiator i (FIG. 1) (50.3 mg) and SStNa (1.56 g) weredissolved in 20 mL of water and degassed under argon for 15 min. Bpy(15.6 mg) and Cu(I)Br (7.2 mg) were then added under stirring at 20° C.After 24 h, the solution was exposed to air and the polymer was purifiedas reported in Example 5. M_(w)=NA; M_(w)/M_(n)=NA. M_(n (NMR))=57 500g/mol.

Example 15 Synthesis of 5-Arm Star Hydroxyethyl Methacrylate/4-StyreneSulfonic Acid Sodium Salt Hydrate Copolymer (69/31 mol % AfterPurification)

The ATRP initiator ii (FIG. 1) (142.6 mg), SStNa (1.55 g) and HEMA(4.616 g) were dissolved in 30 of a methanol/water (8/1) mixture anddegassed under argon for 15 min. Bpy (230.75 ma) and Cu(I)Br (106 mg)were then added under stirring at 20° C. After 1 h of reaction, 10 mL ofwater were added and the solution was then maintained at roomtemperature for 24 h. The corresponding copolymer was finally purifiedas reported in Example 5. M_(w)=85 000 g/mol; M_(w)/M_(n)=1.79.

Example 16 Synthesis of 8.Arm Star Hydroxyethyl Methacrylate/4.StyreneSulfonic Acid Sodium Salt Hydrate Copolymer (75/25 mol % AfterPurification)

The ATRP initiator iv (FIG. 1) (141 mg), SStNa (1.5 g) and HEMA (4.616g) were dissolved in 30 mL of a methanol/water (8/1) mixture anddegassed under argon for 15 min, Bpy (230.8 mg) and Cu(I)Br (106 mg)were then added under stirring at 20° C. After 1 h of reaction, 10 mL ofwater were added and the solution was then maintained at roomtemperature for 24 h. The corresponding copolymer was finally purifiedas reported in Example 5. M_(w)=210 000 g/mol; M_(w)/M_(n)=2.03.

Example 17 Synthesis of 18.Arm Star Hydroxyethyl Methacrylate/4.StyreneSulfonic Acid Sodium Salt Hydrate Copolymer (69/31 mol % AfterPurification)

The ATRP initiator iii (FIG. 1) (153.5 mg), SStNa (1.5 g) and HEMA (4.62g) were dissolved in 30 mL of a methanol/water (8/1) mixture anddegassed under argon for 15 min. Bpy (230.75 mg) and Cu(I)Br (106 mg)were then added under stirring at 20° C. After 1 h of reaction, 10 mL ofwater were added and the solution was then maintained at roomtemperature for 24 h. The corresponding copolymer was finally purifiedas reported in Example 5. M_(w)=206 000 g/mol; M_(w)/M_(n)=2.6.

Example 18 Synthesis of Linear Hydroxyethyl Methacrylate(HEMA)/Sulfopropyl Methacrylate Potassium Salt (SPMAK) Copolymer (86/14mol % After Purification)

The ATRP initiator i (FIG. 1) (100.3 mg), SPMAK (1.85 g) and HEMA (5.62g) were dissolved in 30 mL of methanol and degassed under argon for 15min. Bpy (31.96 mg) and Cu(I)Br (15.1 mg) were then added under stirringat 20° C. After 24 h, the solution was exposed to air and the polymerwas purified as reported in Example 5. M_(w)=NA; M_(w)/M_(n)=NA.Mn_((NMR))=66 500 g/mol.

Example 19 Synthesis of Linear Hydroxyethyl Methacrylate(HEMA)/Sulfopropyl Methacrylate Potassium Salt (SPMAK) Copolymer (83/17mol % After Purification)

The ATRP initiator i (FIG. 1) (102.1 mg), SPMAK (1.90 g) and HEMA (5.62g) were dissolved in 30 mL of methanol and degassed under argon for 15min. Bpy (31.24 mg) and Cu(I)Br (14.34 mg) were then added understirring at 20° C. After 24 h, the solution was exposed to air and thepolymer was purified as reported in Example 5. M_(w)=NA; M_(w)/M_(n)=NA.Mn_((NMR))=84 000 g/mol.

Example 20 Synthesis of Linear Hydroxyethyl Methacrylate/SulfopropylMethacrylate Potassium Salt Copolymer (74/26 mol % After Purification)

The ATRP initiator i (FIG. 1) (100.5 mg), SPMAK (3.75 g) and HEMA (5.622g) were dissolved in 46 mL of a methanol/water (1/1) mixture anddegassed under argon for 15 min. Bpy (32 mg) and Cu(I)Br (15.1 mg) werethen added under stirring at 20° C. After 24 h, the solution was exposedto air and the polymer was purified as reported in Example 5. M_(w)=NA;M_(w)/M_(n)=NA. M_(n (NMR))=119 000 g/mol.

Example 21 Synthesis of Linear Hydroxyethyl Methacrylate/SulfopropylMethacrylate Potassium Salt Copolymer (45/55 mol % After Purification)

The ATRP initiator i (FIG. 1) (100.7 mg), SPMAK (5.64 g) and HEMA (1.752g) were dissolved in 46 mL of a methanol/water (1/1) mixture anddegassed under argon for 15 min. Bpy (32 mg) and Cu(I)Br (15.1 mg) werethen added under stirring at 20° C. After 24 h, the solution was exposedto air and the polymer was purified as reported in Example 5 M_(w)=NA;M_(w)/M_(n)=NA. Mn_((NMR))=108 500 g/mol.

Example 22 Synthesis of Linear Poly(Sulfopropyl Methacrylate Potassium)

The ATRP initiator i (FIG. 1) (100.7 mg) and SPMAK (7.5 g) weredissolved in 46 mL of a methanol/water (1/1) mixture and degassed underargon for 15 min. Bpy (32 mg) and Cu(I)Br (15.1 mg) were then addedunder stirring at 20° C. After 24 h, the solution was exposed to air andthe polymer was purified as reported in Example 5. M_(w)=NA;M_(w)/M_(n)=NA. Mn_((NMR))=120 000 g/mol.

Example 23 Synthesis of 5-Arm Star Hydroxyethyl Methacrylate/SulfopropylMethacrylate Potassium Copolymer (82.4/17.6 mol % After Purification)

The ATRP initiator ii (FIG. 1) (143 mg), SPMAK (1.87 g) and HEMA (4.61g) were dissolved in 60 mL of a methanol/water (8/1) mixture anddegassed under argon for 15 min. Bpy (230.6 mg) and Cu(I)Br (108 mg)were then added under stirring at 20° C. After 24 h, the solution wasexposed to air and the polymer was purified as reported in Example 5.M_(w)=161 000 g/mol; M_(w)/M_(n)=2.4.

Example 24 Synthesis of 8-Arm Star Hydroxyethyl Methacrylate/SulfopropylMethacrylate Potassium Copolymer (81/19 mol % After Purification)

The ATRP initiator iv (FIG. 1) (70.5 mg), SPMAK (0.935 g) and HEMA (2.3g) were dissolved in 60 mL of a methanol/water (8/1) mixture anddegassed under argon for 15 min. Bpy (115.8 mg) and Cu(I)Br (53.9 mg)were then added under stirring at 20° C. After 24 h, the solution wasexposed to air and the polymer was purified as reported in Example 5.M_(w)=227 000 g/mol; M_(w)/M_(n)=2.27.

Example 25 Synthesis of 18-Arm Star HydroxyethylMethacrylate/Sulfopropyl Methacrylate Potassium Copolymer (82.4/17.6 mol% After Purification)

The ATRP initiator iii (FIG. 1) (75.3 mg), SPMAK (0.923 g) and HEMA(2.31 g) were dissolved in 60 mL of a methanol/water (8/1) mixture anddegassed under argon for 15 min. Bpy (115.8 mg) and Cu(I)Br (53.9 mg)were then added under stirring at 20° C. After 24 h, the solution wasexposed to air and the polymer was purified as reported in Example 5.M_(w)=342 000 g/mol; M_(w)/M_(n)=2.28.

Example 26 Assessment of Polymer″Gliadin Binding

The binding selectivity and affinity of gliadin toward the synthesizedpolymers was assessed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SOS-PAGE) using a 15% (w/v) separating gel. Inaddition, the polymers were separately screened for their reactivitytoward control proteins, namely bovine albumin and/or bovine casein.Binding studies were carried out at pH 1.2 and 6.8 using hydrochloricacid and phosphate buffers, respectively. Polymer (80 mg/L) and protein(40 mg/L) were mixed together at pHs 1.2 and 6.8 and incubated for 2 h.The solutions were then centrifuged at 15 000 g for 30 min in order toseparate the insoluble complex from free protein that remained insolution. The supernatant was then analyzed by SDS-PAGE to measure theamount of free protein.

Example 27 Selectivity of Poly(HEMA-Co-SStNa) Binding to Gliadin

The binding affinity of gliadin toward different linearpoly(HEMA-co-SStNa) (synthesis reported in Examples 5 to 10 and 12-14)was assessed by SOS-PAGE as described in Example 26 and compared to thatof albumin and casein (FIG. 3 showing Examples 5 to 10 and 12-13) atintestinal (6.8) and gastric (1.2) pHs. In general, the polymerexhibited greater affinity for gliadin compared to the control proteinsat both pH values. It has to be pointed out that the complexation withcasein was not studied at pH 1.2 due to the insolubility of this proteinunder acidic conditions. As shown by FIG. 3, complexation to gliadincould be modulated by the copolymer composition. FIG. 2 also showsselective binding on SOS-PAGE between gliadin and linearpoly(HEMA-co-SStNa) (Example 10), whereas albumin remained free insolution upon incubating the copolymer with both proteins. The bindingaffinity of gliadin toward the linear poly(HEMA-co-SStNa) polymer ofExample 14 was assessed by SDS-PAGE as described in Example 26 andcompared to that of albumin. Results were as follows: complexation withalbumin at pHs 1.2 and 6.8 was of 78.8% and 11.23%, respectively;complexation with gliadin at pHs 1.2 and 6.8 was of 100% and 71.3%,respectively.

Example 28 Selectivity of Poly(HEMA-co-SPMAK) Composition Binding toGliadin

The binding affinity of gliadin toward different linearpoly(HEMA-co-SPMAK) (synthesis reported in Examples 18 to 22) wasassessed by SDS-PAGE as described in Example 26 and compared to that ofalbumin (FIG. 4) at intestinal (6.8) and gastric pHs (1.2). Lesserbinding to gliadin was observed when the SStNa monomer was replaced bySPMAK especially at pH 6.8 (FIG. 4). Optimal complexation to gliadin wasachieved for SPMAK ratios ranging from 50 to 100 mol %.

Example 29 Effect of Copolymer Structure on Binding to Gliadin

Five, eight and eighteen arms star poly(HEMA-co-SStNa) (Examples 15 to17) and poly(HEMA-co-SPMAK) (Examples 23 to 25) were synthesized usinginitiators derived from glucose, sucrose and cyclodextrin, respectively.Their ability to bind gliadin was compared to their linear counterpart(Examples 9 and 18, respectively). The results are presented in FIGS. 5and 6. For a fixed percentage of SStNa of about 30 mol %, the bindingefficiency of eight arms star poly(HEMA-co-SStNa) was better than thelinear or the other star copolymers (FIG. 5). At a fixed ratio of SPMAKof 17 mol %, no significant difference was observed in the binding ofgliadin to linear or star poly(HEMA-co-SPMAK) (FIG. 6).

Conclusions

Linear and star-shaped random copolymers of HEMA and SStNa or SPMAK wereshown to bind α-gliadin under pH conditions mimicking thegastrointestinal tract.

Example 30 Effect of Copolymer Molecular Weight on Binding to Gliadin

Two different weights linear poly(HEMA-co-SStNa) (Examples 10 and 11)containing about 50% SStNa were tested for the binding of gliadin andalbumin at both pHs 1.2 and 6.8. In this experience, each protein wastested separately. The results are presented in FIG. 9. The binding togliadin and selectivity of binding was found to be influenced by themolecular weight of the polymer.

Example 31 Prevention of Enzymatic Degradation of Gliadin by a Copolymer

Preparation of Peptic-Tryptic Digests of gliadin

The stepwise enzymatic hydrolysis of α-gliadin was performed with pepsin(Sigma P0609; St Louis, Mo., USA) and trypsin (Sigma T1763), bothattached to agarose as well as a-chymotrypsin from bovine pancreas(Sigma C4129). α-Gliadin (10 mg) was dissolved in 5 mL of hydrochloricacid buffer pH=1.2 (10 mM) and pepsin (38 U) was added. The mixture wasmagnetically stirred at 37° C. for 2 hours at which point the pH wasadjusted to 6.8 with 0.1 mol/L NaOH and trypsin (0.75 U) as well asα-chymotrypsin (0.5 U) were added. The digest was centrifuged for 30 minat 20° C. and 6000 g. The gliadin peptides were thereafter collected inthe supernatant and filtered through 0.2 μm GHP filters.

The resulting peptic-tryptic-chymotryptic digest of gliadin was analyzedusing a Waters™ high-performance liquid chromatography HPLC systemequipped with a 1525 Binary pump, a 2487 dual wavelength absorbancedetector, and a Breeze Chromatography Software™ (Waters, Midford,Mass.). Samples were eluted at 36° C. at a flow rate, detectionwavelength, and injection volume of 1 MI/min, 215 nm and 50 μL,respectively. Trifluoroacetic acid was used as an ion pairing agent, andelution was performed with a linear gradient consisting of 100% buffer Ato 100% buffer B spanning over 60 min. Buffer A consisted of 0.1%trifluoroacetic acid, 95% water, and 5% acetonitrile and buffer Bconsisted of 0.1% trifluoroactic acid, 5% water, and 95% acetonitrile. Aportion of each sample supernatant was diluted into water and analysedon a C₁₈ reversed phase column (Waters Novapack™ C18, 60 Å, 4 μm,3.9×300 mm).

Enzymatic Degradation of the Gliadin-Polymer Complex

Poly(HEMA-co-SStNa) (Example 10) (4 g/L) and gliadin (2 g/L) were mixedtogether at pH 2 and incubated for 2 h. Then, the stepwise enzymaticdegradation of gliadin-polymer complex was performed as described above.The effect of the polymeric binder on the degradation of gliadin wasanalysed using HPLC as described above (FIG. 7).

Substantially less degradation products were detected when the gliadinwas complexed to the polymer (FIG. 7).

Example 32 Effect of Polymer on Caco-2 Monolayer Integrity

The effect of poly(HEMA-co-SStNa) (Example 10) on Caco-2 cell monolayerintegrity was assessed and compared to that of PEG (35 kDa) and PVP (58kDa) (FIG. 8). Cells were seeded onto 12-well Transwell® polycarbonatefilters (Corning, Acton, Mass.) at a seeding density of 2.5×10⁵cell/cm². Caco-2 were grown in Dulbecco's modified essential medium(DMEM) supplemented with 10% (v/v) foetal bovine serum, non-essentialamino acid solution (0.1 mM), Hepes buffer pH 7.4 (10 mM) andpenicillin-streptomycin (eq. 100 U/mL and 100 μg/mL). Medium wasrefreshed every 72 h. Cells were cultured for 21-28 days at 37° C., 5%CO₂ to form a differentiated monolayer prior to the experiments.Toxicity studies were performed in complete DMEM medium. Transepithelialelectrical resistance (TEER) readings were taken at pre-determinedtime-points using a Millicell™ Electrical Resistance System (MilliporeCorp. Bedford, Mass.) with a single electrode (World PrecisionInstruments, Sarasota, Fla.).

In both the Poly(HEMA-co-SStNa) and control polymers (PVP, PEG), theTEER measured after 24 hours showed a reduction of 10% of the initialvalue (FIG. 8). These results indicate that Poly(HEMA-co-SStNa) do notseem to strongly perturb the integrity of the Caco-2 cell monolayer.

Example 33 In Vivo Testing of Effect of Polymeric Binder on Reduction ofToxicity of Gliadin and Gliadin Degradation Products

The ability of the polymer to reduce the toxicity of gluten is evaluatedin vivo by measuring the immune response of animals that have beensensitized to gluten or its degradation products. The immune response ismeasured in transgenic mice expressing HLA-DQ8 (24) following oraladministration of gluten or its degradation products in the presence orabsence of polymeric binder.

Example 34 Incorporation of Polymeric Binder in Food

The polymeric binder may be incorporated into gluten-containing fooddirected to individuals affected by celiac disease. The polymeric binderin such food may then counteract the deleterious effects of the glutencontained in the food when it is swallowed. Without being so limited,such food includes ready-cooked dishes, cereals, baked goods such bread,pastry, pies, cakes, muffins, cookies etc. Such food may incorporate thepolymeric binder in a concentration of 0.01% to 10% (w/w). The polymericbinder can also be incorporated into non gluten-containing food forconsumption in a meal containing gluten-containing food. Without beingso limited, such non gluten-containing food includes spreads such ascheese, jams, butter or any food that can be eaten on or withgluten-containing food.

Although the present invention has been described hereinabove by way ofspecific embodiments thereof, it can be modified, without departing fromthe spirit and nature of the subject invention as defined in theappended claims.

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The invention claimed is:
 1. Food comprising a pharmaceuticallyeffective amount of a polymeric binder including a high molecular weightsynthetic linear copolymer, the synthetic linear copolymer comprising alinear copolymer of hydroxyethyl methacrylate (HEMA) and 4-styrenesulfonic acid or a salt thereof, wherein said linear copolymer has amolar percentage ratio of HEMA:4-styrene sulfonic acid or a salt thereoffrom between about 82.4:17.6 mol % to about 28:72 mol %, and a processedfood.
 2. The food of claim 1, wherein the food is a gluten-containingfood.
 3. The food of claim 2, wherein the food is bread.
 4. The food ofclaim 1, wherein the synthetic linear copolymer comprises a copolymer ofHEMA and SStNa hydrate.
 5. The food of claim 4, wherein the copolymer ofHEMA and SStNa is linear HEMA/SStNa, wherein the HEMA/SStNa ratio is51.5/48.5 mol %.
 6. The food of claim 4, wherein the copolymer of HEMAand SStNa is linear HEMA/SStNa, wherein the HEMA/SStNa ratio is 43/57mol %.
 7. The food of claim 4, wherein the synthetic linear copolymer ofHEMA and SStNa contains about 50% SStNa.
 8. The food of claim 1, whereinthe synthetic linear copolymer has a backbone constituted ofnon-hydrolysable covalent bonds.
 9. The food of claim 1, wherein thesynthetic linear copolymer is able to form electrostatic bonds at a pHlower than the isoelectric point of gluten and peptides derived from thedegradation of gluten.
 10. The food of claim 1, wherein the syntheticlinear copolymer is able to form hydrophobic interactions with gluten orpeptides derived from the degradation of gluten.
 11. The food of claim1, wherein the synthetic linear copolymer is able to form hydrogenbonds.
 12. The food of claim 1, wherein the synthetic linear copolymeris able to specifically bind to gluten or peptides derived from thedegradation of gluten in the gastrointestinal tract.
 13. The food ofclaim 1, wherein the synthetic linear copolymer is able to bind togluten or peptides derived from the degradation of gluten in theintestinal tract.